CN101776642A - Mass spectrometric detection method for ca-dependent secretory protein 1 in human serum - Google Patents

Mass spectrometric detection method for ca-dependent secretory protein 1 in human serum Download PDF

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CN101776642A
CN101776642A CN201010105552A CN201010105552A CN101776642A CN 101776642 A CN101776642 A CN 101776642A CN 201010105552 A CN201010105552 A CN 201010105552A CN 201010105552 A CN201010105552 A CN 201010105552A CN 101776642 A CN101776642 A CN 101776642A
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magnetic bead
mass
peak
human serum
secretory protein
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CN101776642B (en
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沈锡中
薛如意
刘韬韬
董玲
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Zhongshan Hospital Fudan University
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Zhongshan Hospital Fudan University
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Abstract

The invention relates to a mass spectrometric detection method for ca-dependent secretory protein 1 in human serum, which is characterized in that the mass spectrometric detection method comprises the following steps: taking human serum and using magnetic bead attachment protein for obtaining magnetic bead enrichment liquid; adding the magnetic bead enrichment liquid into a matrix solution; carrying out the matrix-assisted laser dissociation ionization flight time mass spectrometric detection according to the following conditions: a first ion source voltage: 20kV; a second ion source voltage: 18.6kV; crystal voltage: 7.6kV; pulse ion time: 320ns; polarity: positive; matrix inhibition mode: gate control; gate control strength: maximum: suppression high limit: 600Da; detector: 1400-1500 V; sample rate: 1.00Gs/s; electrical acquisition: enhancing, 100mV; real-time fluency: high; laser frequency: 20Hz; laser attenuation: closing 73% and ranging 20%; mass to charge ratio is 5336 plus or minus 1; and the peak flowing is the ca-dependent secretory protein 1. The method has the advantages of short time and high sensitivity.

Description

The Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum
Technical field
The present invention relates to the Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum, be specifically related to a kind of employing enrichment with magnetic bead associating ground substance assistant laser parsing/ionization time of flight mass spectrometry (matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry, MALDI-TOF MS) method of screening ca-dependent secretory protein 1 (caps1) from human serum belongs to the method for protein detection technical field.
Background technology
(Calcium-dependent secretion activator 1 CAPS-1) is a kind of calbindin to ca-dependent secretory protein 1, can promote neurotransmitter, neuropeptide vesica to be transported to cell membrane and further discharge secretory granules.The main detection method of caps1 comprises protein immunization marking method, enzyme linked immunosorbent assay at present, and their shortcoming is consuming time, effort, and the insufficient sensitivity height is unsuitable for the screening of extensive sample.
Summary of the invention
The objective of the invention is to overcome the problems referred to above, the Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum is provided.
In order to achieve the above object, technical scheme of the present invention provides the Mass Spectrometer Method method of ca-dependent secretory protein 1 in a kind of human serum, it is characterized in that, concrete steps are:
The first step: take people's whole blood, solidified under 4 degrees centigrade 1 hour, centrifugal, get upper serum, be kept in-80 degrees centigrade of refrigerators, weak cation is exchanged magnetic bead or the affine copper ion magnetic bead of immune metal to be combined liquid and mixes in thin-walled PCR pipe with magnetic bead, add serum sample, incubated at room 5min places thin-walled PCR pipe and carries out magnetic bead in the magnetic bead separation vessel and separate, remove supernatant, after the washing of last magnetic bead usefulness magnetic bead cleansing solution, add the magnetic bead eluent, thin-walled PCR pipe is placed carry out the magnetic bead separation in the magnetic bead separation vessel, obtain eluent, eluent is mixed obtaining enrichment with magnetic bead liquid with magnetic bead stabilizing solution;
Second step: the enrichment with magnetic bead liquid that the first step is obtained adds matrix solution, carries out the ground substance assistant laser ionization time of flight mass spectrometry that dissociates by following condition and detects: first ion gun voltage: the 20kv; Second ion gun voltage: the 18.6kv; Crystal voltage: 7.6kv; The pulse ion time: 320ns; Polarity: just; Matrix suppression mode: gate; Gate intensity: maximum; Suppress high limit: 600Da; Detecting device: 1400-1500v; Sample rate: 1.00Gs/s; Electronics obtains: strengthen 100mv; Smooth in real time: height; Laser frequency: 20Hz; Laser attenuation: close 73%, scope 20%; The peak examination criteria: signal to noise ratio (S/N ratio) is greater than 5, and the 2Da peak width is filtered, at most peak number 200;
The 3rd step: analyze the mass spectrogram that second step obtained, observe mass-to-charge ratio is whether 5336 ± 1 places have the peak in the mass spectrogram,, then contain ca-dependent secretory protein 1 in the human serum in the serum if the peak is arranged.
Advantage of the present invention is: this method is selected for use fast and the responsive ground substance assistant laser ionization time of flight mass spectrometry that dissociates detects serum caps1, belong to the proteomics research category, ground substance assistant laser desorption ionization flight time mass spectrum is a very effective instrument finding and identify aspect the albumen of disease association or the polypeptide, be used to seek multiple disease specific sign, the method for this technical tie-up magnetic particle enrichment haemocyanin more fast, effectively and accurately.This hypersensitivity and repeatable technology based on magnetic particle is allowed to condition at the protein science research field and takes the course of its own.The each detection of this method only needs tens of minutes time, the enrichment and the detection of serum caps1 albumen have been finished, simple to operate with respect to traditional protein immunization marking method, enzyme linked immunosorbent assay (consuming time more than 8 hours), save time, be convenient to the detection of sample in enormous quantities; Because of not relying on many restrictions that biochemical reaction is not subjected to reaction conditions, repeatability is high; The detection sensitivity of classic method is a Gamma Magnitude in addition, and the detection sensitivity Da Nake level of this method is significantly higher than traditional method.
Description of drawings
Fig. 1 is a through the human serum Mass Spectrometer Method figure behind the enrichment with magnetic bead.
Embodiment
Specify the present invention below in conjunction with embodiment.
Embodiment
1. main agents:
Weak cation exchange magnetic bead MB-WCX kit (Bruker Daltonik GmbH, Germany),
Immunity metal affine copper ion magnetic bead MB-IMAC Cu kit (Bruker Daltonik GmbH, Germany)
Alpha-cyano-4-hydroxycinnamic acid α-Cyano-4-hydroxycinnamic acid (HCCA) (sigma, the U.S.)
Acetone (sigma, the U.S.)
Ethanol (sigma, the U.S.)
AnchorChip TMTarget (Bruker Daltonik GmbH, Germany)
2. consumptive material and instrument:
Vacuum test tube (BD Vacutainer TM, the U.S.)
Low temperature refrigerator (SANYO, Japan)
Vortex mixed instrument (H101, last Haikang standing grain photoelectric instrument company limited)
Constant temperature supercentrifuge (Z400K, HERMLE, Germany)
Magnetic bead separation vessel (Bruker Daltonik GmbH, Germany)
Autoflex II ground substance assistant laser dissociating ions time-of-flight mass spectrometry (Bruker Daltonik GmbH, Germany)
LC/MS/MS combined instrument (u.s.a. applied biosystem company, Shanghai)
Other: syringe, Eppendorf pipe, application of sample rifle, rifle are first-class
3. the collection of biological sample:
Select BD Vacutainer for use TMAfter vacuum test tube (yellow skull) is taked people's whole blood, solidified 1 hour under 4 degree, press 3000rpm 10min altogether, get upper serum, be kept in the Eppendorf pipe stand-by in-80 degree refrigerators with hydro-extractor.
4. serum sample pre-treatment:
(1) on the vortex mixed instrument, weak cation exchanged MB-WCX magnetic bead solution or the thorough mixing 1min of the affine copper ion MB-IMACCu of immune metal magnetic bead solution.
(2) 10 μ l MB-WCX magnetic beads are transferred in the thin-walled PCR pipe of a standard in conjunction with liquid and 10 μ l WCX magnetic bead solution, up and down the pressure-vaccum mixing.(or with 50 μ l MB-IMAC Cu magnetic beads in conjunction with liquid pre-service 5 μ l MB-IMAC Cu magnetic bead solution, thin-walled PCR pipe is put into the magnetic bead separation vessel, thin-walled PCR pipe is moved 10 times between adjacent holes, assemble magnetic bead in tube wall 20s, carefully remove supernatant with rifle, repeat secondary, MB-IMAC Cu magnetic bead is resuspended in 20 μ l MB-IMAC Cu magnetic beads in conjunction with in the liquid).
Add 5 μ l human serum samples, pressure-vaccum is 5 times up and down, mixing liquid.Incubated at room 5min.The PCR pipe is placed in the magnetic bead separation vessel, pipe is moved forward and backward 3 times after, assemble magnetic bead in tube wall 1min, carefully remove supernatant with rifle.
Xiang Guanzhong adds 100 μ l magnetic bead cleansing solutions, moves around 10 times before and after the PCR pipe is in the magnetic bead separation vessel, notes moving of magnetic bead.Assemble magnetic bead 20s on tube wall, carefully remove supernatant with rifle.Repeat this step twice.
Add 5 μ l MB-WCX magnetic bead eluents (or 10 μ l MB-IMAC Cu magnetic bead eluents), 10 times magnetic bead is dissolved from tube wall, wait for 5min by strong pressure-vaccum up and down.
Pipe is put into the magnetic bead separation vessel, assemble magnetic bead 2min on tube wall, the supernatant that will contain the wash-out molecule shifts in the clean pipe.In eluent, add 4 μ l magnetic bead stabilizing solution, the strong mixings of pressure-vaccum up and down.
The sample of 1 μ l wash-out is added the matrix solution (HCCA solution, it is at ethanol: acetone is that concentration is 0.3g/l in 2: 1 the solution) of 10 μ l, put 1 μ l to MALDI-TOF mass spectrum target.
5. mass spectrum condition:
First ion gun voltage: the 20kv; Second ion gun voltage: the 18.6kv; Crystal voltage: 7.6kv; The pulse ion time: 320ns; Polarity: just; Matrix suppression mode: gate; Gate intensity: maximum; Suppress high limit: 600Da; Detecting device: 1400-1500v; Sample rate: 1.00Gs/s; Electronics obtains: strengthen 100mv; Smooth in real time: height; Laser frequency: 20Hz; Laser attenuation: close 73%, scope 20%.The peak examination criteria: signal to noise ratio (S/N ratio) is greater than 5, and the 2Da peak width is filtered, at most peak number 200.
6. testing result:
As shown in Figure 1, the human serum Mass Spectrometer Method figure for behind the portion process enrichment with magnetic bead is analyzed as follows:
Sequence number Mass-to-charge ratio Peak area
??1 ??1206.852 ??233.2806
??2 ??1234.907 ??164.5131
??3 ??1308.592 ??366.7388
??4 ??1316.401 ??519.6103
??5 ??1321.026 ??344.6194
??6 ??1329.471 ??1089.298
??7 ??1338.959 ??435.7691
??8 ??1349.922 ??221.7832
??9 ??1364.439 ??212.3075
??10 ??1385.022 ??9.148874
??11 ??1430.335 ??294.3772
Sequence number Mass-to-charge ratio Peak area
??12 ??1465.247 ??379.206
??13 ??1471.953 ??310.7988
??14 ??1545.199 ??78.50821
??15 ??1619.369 ??1522.463
??16 ??1631.186 ??2146.741
??17 ??1717.474 ??396.6772
??18 ??1725.901 ??103.1431
??19 ??1738.477 ??138.6453
??20 ??1746.532 ??684.2241
??21 ??1763.722 ??960.641
??22 ??1777.051 ??548.2078
??23 ??1808.028 ??262.3039
??24 ??1813.866 ??498.4405
??25 ??1829.415 ??381.9238
??26 ??1867.389 ??236.5915
??27 ??1888.154 ??3469.796
??28 ??1945.111 ??7614.77
??29 ??1979.032 ??1031.452
??30 ??2046.854 ??665.103
??31 ??2081.432 ??7881.219
??32 ??2091.556 ??1892.181
??33 ??2104.803 ??10728.67
Sequence number Mass-to-charge ratio Peak area
??34 ??2209.504 ??3756.636
??35 ??2294.414 ??2939.391
??36 ??2344.978 ??1147.049
??37 ??2353.17 ??103.2464
??38 ??2364.338 ??1158.016
??39 ??2404.817 ??773.1838
??40 ??2420.323 ??1115.697
??41 ??2481.959 ??731.6669
??42 ??2559.922 ??9919.333
??43 ??2611.364 ??190.0135
??44 ??2623.927 ??259.4034
??45 ??2630.597 ??365.6349
??46 ??2645.672 ??4443.032
??47 ??2660.64 ??77700.35
??48 ??2725.39 ??1544.949
??49 ??2739.036 ??941.1075
??50 ??2769.317 ??14584.53
??51 ??2862.939 ??31985.49
??52 ??2883.266 ??9414.285
??53 ??2902.075 ??3810.259
??54 ??2932.038 ??24462.84
??55 ??2951.82 ??41541.18
Sequence number Mass-to-charge ratio Peak area
??56 ??3192.049 ??21752.23
??57 ??3222.155 ??2963.911
??58 ??3224.215 ??2963.911
??59 ??3241.183 ??46399.18
??60 ??3263.096 ??40800.38
??61 ??3447.513 ??603.8208
??62 ??3505.064 ??1879.339
??63 ??3523.206 ??4182.684
??64 ??3538.167 ??1750.341
??65 ??3586.819 ??664.058
??66 ??3616.573 ??126.8505
??67 ??3636.45 ??1764.688
??68 ??3678.28 ??2093.34
??69 ??3680.634 ??2093.34
??70 ??3697.631 ??795.8046
??71 ??3726.096 ??1776.15
??72 ??3952.973 ??8315.439
??73 ??4054.517 ??11959.5
??74 ??4073.242 ??4533.026
??75 ??4090.748 ??56336.94
??76 ??4121.616 ??2703.324
??77 ??4154.886 ??1807.75
Sequence number Mass-to-charge ratio Peak area
??78 ??4168.399 ??3301.79
??79 ??4209.553 ??240623.3
??80 ??4269.454 ??10226.33
??81 ??4526.278 ??1213.465
??82 ??4643.493 ??67880.99
??83 ??4672.354 ??14847.94
??84 ??4787.175 ??5741.917
??85 ??4915.494 ??1385.752
??86 ??4936.063 ??834.1923
??87 ??4963.46 ??41928.48
??88 ??5065.108 ??8760.362
??89 ??5290.733 ??806.0496
??90 ??5336.44 ??130785.5
??91 ??5398.947 ??1636.666
??92 ??5479.533 ??504.1858
??93 ??5523.46 ??4029.33
??94 ??5904.121 ??401500.5
??95 ??5966.6 ??65.17127
??96 ??6089.927 ??2243.643
??97 ??6630.462 ??12355.21
??98 ??7765.259 ??36881.82
??99 ??9288.768 ??196286.3
Sequence number Mass-to-charge ratio Peak area
??100 ??9347.858 ??5288.739
Mass-to-charge ratio is the peak that the peak at 5336.44 places is ca-dependent secretory protein 1 in the last table.
7, utilize the liquid chromatography mass GC-MS further to identify:
Enrichment with magnetic bead liquid is identified with the liquid chromatography mass GC-MS, with the MASCOT program mass spectrometric data is carried out online international albumen parameter (International Protein Index, IPI) database (http://www.ebi.ac.uk) inquiry.
Wherein, liquid chromatography-mass spectrography/mass spectroscopy device is connected the LTQ-Orbitrap mass spectrum and is constituted by the nanometer stream liquid chromatography pump of two LC-20AD, SIL-20AC automatic sampler, a LC-20AB miniflow liquid chromatography pump.Sample fills in the CAPTRAP post, flow velocity 50ul/ branch enters the C18 reversed-phase column and carries out liquid phase separation after 3 minutes, flow velocity 500nl/ branch, moving phase is 5% acetonitrile and 0.1% formic acid (A mutually, filling phase), 95% acetonitrile and 0.1% formic acid (B mutually), B phase linear gradient 5%-45% (60 minutes).Sample after the separation enters mass spectrometer by ADVANCE30um silicon dioxide interface end, spray voltage 1kv, and 180 ℃ of temperature, data rely on pattern, each complete scanning of the mass spectrum mass range 400-2000Da, resolving power 100000.The peptide section is at LTQ certain applications helium collision induced dissociation, and mark is dissolved from energy value 35%.The peak that dissociates of band 2+ and 3+ is selected does mass spectrum/mass spectrophotometry.Select for use BioWorks 3.3.1 sp1 software that IPI human protein database is carried out on-line search, condition: 2 possible error sites, the polypeptide mass deviation allows for 10ppm, and the fragment mass deviation allows for 1.00Da.Mass-to-charge ratio is that 5336 ± 1 material peak is accredited as caps1 albumen.

Claims (1)

1. the Mass Spectrometer Method method of ca-dependent secretory protein 1 in the human serum is characterized in that concrete steps are:
The first step: take people's whole blood, solidified under 4 degrees centigrade 1 hour, centrifugal, get upper serum, be kept in-80 degrees centigrade of refrigerators, weak cation is exchanged magnetic bead or the affine copper ion magnetic bead of immune metal to be combined liquid and mixes in thin-walled PCR pipe with magnetic bead, add serum sample, incubated at room 5min places thin-walled PCR pipe and carries out magnetic bead in the magnetic bead separation vessel and separate, remove supernatant, after the washing of last magnetic bead usefulness magnetic bead cleansing solution, add the magnetic bead eluent, thin-walled PCR pipe is placed carry out the magnetic bead separation in the magnetic bead separation vessel, obtain eluent, eluent is mixed obtaining enrichment with magnetic bead liquid with magnetic bead stabilizing solution;
Second step: the enrichment with magnetic bead liquid that the first step is obtained adds matrix solution, carries out the ground substance assistant laser ionization time of flight mass spectrometry that dissociates by following condition and detects: first ion gun voltage: the 20kv; Second ion gun voltage: the 18.6kv; Crystal voltage: 7.6kv; The pulse ion time: 320ns; Polarity: just; Matrix suppression mode: gate; Gate intensity: maximum; Suppress high limit: 600Da; Detecting device: 1400-1500v; Sample rate: 1.00Gs/s; Electronics obtains: strengthen 100mv; Smooth in real time: height; Laser frequency: 20Hz; Laser attenuation: close 73%, scope 20%; The peak examination criteria: signal to noise ratio (S/N ratio) is greater than 5, and the 2Da peak width is filtered, at most peak number 200;
The 3rd step: analyze the mass spectrogram that second step obtained, observe mass-to-charge ratio is whether 5336 ± 1 places have the peak in the mass spectrogram,, then contain ca-dependent secretory protein 1 in the human serum in the serum if the peak is arranged.
CN 201010105552 2010-02-04 2010-02-04 Mass spectrometric detection method for ca-dependent secretory protein 1 in human serum Expired - Fee Related CN101776642B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109142501A (en) * 2015-03-17 2019-01-04 北京毅新博创生物科技有限公司 Full-automatic polypeptide extracts flight time mass spectrum detector
CN114324557A (en) * 2021-12-03 2022-04-12 融智生物科技（青岛）有限公司 Zeta-globin detection method based on MALDI-TOF MS
CN114324557B (en) * 2021-12-03 2024-05-10 融智生物科技（青岛）有限公司 Zeta-globin detection method based on MALDI-TOF MS

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109142501A (en) * 2015-03-17 2019-01-04 北京毅新博创生物科技有限公司 Full-automatic polypeptide extracts flight time mass spectrum detector
CN114324557A (en) * 2021-12-03 2022-04-12 融智生物科技（青岛）有限公司 Zeta-globin detection method based on MALDI-TOF MS
CN114324557B (en) * 2021-12-03 2024-05-10 融智生物科技（青岛）有限公司 Zeta-globin detection method based on MALDI-TOF MS

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